(a) Field of the Invention
The present invention relates to a method for quantitative analysis of peptide interactions, more specifically to an interaction between fluorescein-labeled HIF-1α (alpha) C-terminal peptides and cAMP-responsive element binding protein (CBP) or p300 protein, and a method of screening inhibitors against interactions between HIF-1α peptides and CBP or p300 using the above method, namely a fluorescence polarization (FP)-based binding assay.
(b) Description of the Related Art
It is essential for existence that mammalian cells possess the ability to sense and respond to changes in oxygen levels, and this ability plays an important role in development and physiological processes as well as in human diseases such as angiogenesis, myocardial ischemia, cerebral ischemia, pulmonary hypertension, and various types of cancer. The responses to decreased oxygen level (hypoxia) are mediated by the master transcription factor, HIF-1 (hypoxia inducible factor-1).
HIF-1 (hypoxia inducible factor-1) is composed of α and β subunits, wherein the β subunit is always stably expressed, but HIF-1α (α subunit) is stabilized under a low oxygen condition (hypoxia), and degraded by proteosome under a normal oxygen condition (normoxia). Under hypoxia, the stabilized HIF-1α forms a protein complex that is combined by interactions between HIF-1α and transcription coactivators CBP (cAMP-response element-binding protein) or p300 in the nucleus, and subsequently activates transcription of target genes through binding with a hypoxia-response element (HRE) that exists at the promoter of target genes (Lando, D., et al., Genes. Dev., 16: 1466-14, 2002).
Because these CBP and p300 coactivators control the expression of the specific genes that are correlated with cell growth, differentiation, and homeostasis maintenance through operating in the end point of various signal transductions, controlling interactions between HIF-1α and CPB or p300 proteins is useful for not only treatment of ischemic diseases as to coronary insufficiency, brain ischemia, and blood ischemia, but also for research of cancer therapy involved with inhibiting blood vessel formation.
The CBP or p300 protein has a cysteine-/histidine-rich 1(CH1) domain containing Zn2+ binding factor, wherein the CH1 domain interacts with the C-terminal transactivation domain (C-TAD) of HIF-1α (Kung, A. L., et al., Nature Medicine, 6: 1335-1340, 2000). Also, it has been known that each of complex structures formed by interactions between 331-418th amino acid regions of p300 protein and 792-824th amino acid peptides of HIF-1α, and between 345-439th amino acid regions of CBP protein and 776-826th amino acid peptides of HIF-1α, were revealed by NMR research (Dames, S. A., et al., PNAS, 99: 5271-5276, 2002). It can be determined from these complex structures that the CH1 is stabilized by various hydrophobic and ionic interactions, because it is operating as a scaffold for the folding of the C-terminal domain of HIF-1α. Also, unlike NMR analysis which uses peptides at a high concentration of several mM, the present invention exploited a binding analysis that uses the amino acid regions of HIF-1α that are able to sufficiently bind even at low concentration of μM units.
The expression of HIF-1 dependent target genes is controlled by two different oxygen-dependent mechanisms, proline hydroxylation and asparagine hydroxylation. The hydroxylation of proline residues in the ODD (oxygen-dependent domain) of HIF-1α by HIF-1α-specific prolyl hydoryxlases induces HIF-1α destruction, but the hydroxylation of asparagines diminishes the expression of HIF-1 dependent target genes by inactivating the interaction between the C-TAD of HIF-1α and CBP or p300.
Initially, FIH-1 (Factor-inhibiting HIF-1) protein (Mahon, P. C., et al., Genes Dev., 15: 2675-2686, 2001) was known to hinder HIF-1; however, it was revealed thereafter that it has an oxygen sensor feature that participates in the control of HIF-1α (Lando, D., et al., Gene Dev., 16: 1466-1471, 2002). Namely, the FIH-1 belongs to 2-oxoglutarate and iron-dependent dioxygenase protein (Safran, M., et al., J. Clin. Invest., 111: 779-783, 2003), and hydroxylates Asn-803 in the C-TAD of HIF-1α, thereby disrupting the interaction of HIF-1α with CBP or p300 and blocking the HIF-mediated transactivation (Lando, D., et al., Science, 295: 858-861, 2002).
In addition to asparagine hydroxylation for the transcriptional activity of HIF-1α toward target genes, posttranslational modifications of other residues in C-TAD have been proposed to influence the fine-turning of HIF-1α function (Brahimi-Horn., et al., Cell, Signal., 17: 1-9, 2005). One of representative mechanisms for HIF-1α transcriptional activity control is phosphorylation of transcriptional factors (Holmberg, C. I., et al., Trends Biochem. Sci., 27: 619-627, 2002), namely direct phosphorylation of HIF-1α under normoxia and hypoxia has been reported (Brahimi-Horn, C., et al., Cellular Signaling, 17: 1-9, 2005).
Phosphorylation of HIF-1α can also modulate its transcriptional activity. The activation of mitogen-activated protein kinase (MAPK) induced by hypoxia has been suggested to phosphorylate HIF-1α, which appears to increase the interaction between HIF-1α and p300 protein, but is not correlated with its transcriptional activity (Sang, N., et al., J. Biol. Chem., 278: 14013-14019, 2003). However, the phosphorylation sites responsible for nuclear accumulation of HIF-1α have been identified to reside far from the C-TAD (Mylonis, I., et al., J. Biol. Chem., 281: 33095-33106, 2006). On the other hand, Thr-796 in C-TAD has been indicated as a candidate for phosphorylation possibly by casein kinase 2 (CK2) (Mottet, D., et al., Int. J. Cancer, 117: 764-774, 2005). Despite the abundance of data indicating transcription activity affected by posttranslational modifications of the C-TAD of HIF-1α, disputable information is also available for the direct effects of such modifications on the binding between HIF-1α C-TAD and p300 or CBP proteins.
In addition, it has been reported that NO (nitric oxide) increases or decreases the stability of HIF-1α depending on the cell type and NO concentration (Bilton, R. L., et al., Eur. J. Biochem., 270: 791-798, 2003). In addition, S-nitrosylation of Cys-800 of HIF-1α has been reported to increase HIF-1α transcriptional activity possibly by enhancing its p300 binding (Yasinska, I. M., et al., FEBS Lett., 549: 105-109, 2003), but some conflicting results have also been reported (Brahimi-Horn, C., et al., Cell, Signal., 17: 1-9, 2005).
Until now, various biochemical or immunological methods have been used for analysis of interactions between HIF-1α and CBP or p300. First of all, a two-hybrid assay has been used as a method that measures interactions between HIF-1α and CBP or p300, and in more detail, a method for measuring the transcriptional activity by binding of two proteins, wherein the proteins were manufactured by fusing DNA binding domain (DBD) of yeast GAL4 transcription factor with CBP or p300, and its transcriptional activation domain (TAD) with HIF-1α, respectively.
In another method, one protein among HIF-1α and CBP or p300 is labeled with easily detectable material and the other protein is fixed in the solid support, and then the interaction of the two proteins is measured.
Another method is based on coimmunoprecipitates using the antibody that recognizes the specific region of HIF-1α to identify CBP or p300 and HIF-1α binding. However, these methods have many shortcomings in that massive reagents are demanded, that the process is complicated, that the test time is long, and that a radioactive isotope must be used.
Additionally, there is an indirect method, namely a reporter assay that reveals the transcriptional activity by the interaction between HIF-1α and hypoxia response element (HRE) on target genes after HIF-1α binding to CBP or p300 proteins. One example is a method using the Epo-Luciferase (Kung, A. L., et al., Nature Medicine, 6: 1355-1340, 2000).
Therefore, a simpler measuring method for quantitative analysis of the interactions between HIF-1α and CBP or p300 has been in demand, because the biochemical, immunological, or radioactive isotope-labeling methods as described above have many shortcomings in that massive reagents are demanded, that the process is complicated, that the test time is long, and that the radioactive isotope must be used.
As a result, an analyzing method using 96-well plates that is able to observe interactions between HIF-1α and CBP or p300 in a relatively short time without use of radioactive materials was developed (Kung, A. L., et al., Cancer Cell, 6: 33-43, 2004). This method is useful for screening chemical compounds inhibiting interactions between HIF-1α and CBP or p300, but has shortcomings of the requirement of using a plate coated with expensive avidin, europium-fused anti-GST antibody, and time-resolved fluorescence spectrometer.